Comparative Proteomics Analysis of the Post‐Mitochondrial Fraction (S9 Fraction) of Human Lens‐Free Whole Eye and Liver

Abstract

Background The increasing incidence of ocular diseases has accelerated research into therapeutic interventions for the eye. The knowledge of key metabolic mechanisms in the eye is vital for the successful ophthalmic drug discovery. The lens-free whole-eye post-mitochondrial fraction(S9 fraction) is one of the most widely used in vitro system for ocular metabolism studies during early drug discovery stage. As of now, no report of a comprehensive proteomics investigation exists for the pooled ocular S9 fraction. The objective of this study was to explore the human ocular S9 proteome and compare it with the human liver S9 proteome. Methods The global proteomics study was performed on the pooled ocular (n=11) and hepatic (n=50) S9 fraction samples in data-dependent acquisition mode using an optimized protocol. The samples were acquired in triplicate to minimize the technical variability. The acquired proteomics data were analyzed using Maxquant with 1% false discovery rate. A FASTA file containing whole human proteome, was used as the reference sequence for the search in the Andromeda database search tool. Additionally, another FASTA file containing 1187 proteins involved in the metabolism of endobiotics and xenobiotics was also searched to specifically identify the metabolic enzymes in both organs. The proteins were quantified using a generic label free quantification method (MaxLFQ) in Maxquant software. The metabolic enzymes common to both eye and liver S9 fractions were compared to determine the relative abundance values based on the resulting MaxLFQ intensities. The cut off for the differentially expressed proteins between the eye or liver was either <0.5 or >2. Results and Conclusion A total of 269 proteins (including 23 metabolic enzymes) were detected exclusively in the pooled eye S9, against 648 proteins in the liver S9 (including 174 metabolic enzymes), whereas 424 proteins (including 94 metabolic enzymes) were detected in both the organs. Among the common metabolic enzymes, 18 were predominant in the eye (Figure 1), 43 were predominant in the liver, while rest 33 were comparable in both samples. The major DMEs, viz., Cytochrome P450s, UDP-glucuronosyltransferases, and sulfotransferases, were not detected in the eye S9 fraction, instead isoforms of alcohol dehydrogenases, aldehyde dehydrogenases, carbonic anhydrases, aldo-keto reductases, esterases, epoxide hydrolases, and glutathione S-transferases were in abundance. This information bears great significance in gaining deeper insights on metabolic and bioactivation potential of both systemically as well as topically administered ophthalmic drugs as well as in the endobiotics metabolism within the eye. This investigation provides foundation for the subsequent targeted proteomic, genomic, and functional studies to explore the effects of sex, age, genotype and disease conditions across the populations.